<p>Dissolved oxygen (DO) variability at diel and tidal timescales reflects the balance between physical forcing and biological production in shallow estuaries. Here, we quantify how river flow, phytoplankton biomass, and light availability are associated with diel- and tidal-frequency DO variability (variance in DO at diel and semidiurnal frequencies) along the fresh-to-mesohaline gradient of a shallow estuary, and how these relationships differ across pre- and post-eutrophication periods. We analyzed a high-frequency DO record (2003–2021) from multiple salinity zones in the nutrient-enriched Patuxent River Estuary, a tributary of Chesapeake Bay, supplemented with historical data from the 1960s (pre-eutrophication) and 1990s (post-eutrophication). In the upper estuary, river flow explained up to 40% of diel-frequency DO variability, with higher flow reducing diel variability through enhanced flushing and lower phytoplankton biomass. In the lower estuary, river flow explained 35% of diel- and 25% of tidal-frequency DO variability, reflecting coupled effects of biological production and tidal mixing. Estuary-wide, chlorophyll-a accounted for 14% of diel-frequency DO variability, and turbidity explained 10% of variance. Across eutrophication periods, relationships between DO variability and its physical and biological drivers remained consistent, but increases in nutrient and freshwater inputs coincided with higher chlorophyll-a, amplified DO variability, and longer hypoxia. Our results show that greater freshwater input is associated with increased DO variability and hypoxia across much of the lower estuary, but reduced DO variability in the upper estuary. These findings display that watershed inputs have contrasting, location-specific impacts on DO and chlorophyll-a and inform management programs targeting these water quality variables.</p>